CN106755273A - A kind of selective medium for detecting swine fever brickpox swine plague trigeminal live vaccine antigen bacterial content and its preparation method and application - Google Patents
A kind of selective medium for detecting swine fever brickpox swine plague trigeminal live vaccine antigen bacterial content and its preparation method and application Download PDFInfo
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- CN106755273A CN106755273A CN201710183958.2A CN201710183958A CN106755273A CN 106755273 A CN106755273 A CN 106755273A CN 201710183958 A CN201710183958 A CN 201710183958A CN 106755273 A CN106755273 A CN 106755273A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
Abstract
The invention belongs to the microbiology and production field of animal medicine, a kind of selective medium for detecting swine fever brickpox swine plague trigeminal live vaccine antigen bacterial content and its preparation method and application is specifically disclosed.The selecting property culture medium includes：The 40mg/ml of TSA (tryptose soya agar) 29, cow's serum 3%~5%, the μ g/mL of kanamycins 20~80, appropriate aseptic double-distilled water.The selective medium that the present invention is provided, constitutes component and simple structure, prepares convenient easy to operate；The assay method of the utilization selective medium that the present invention is provided can be distinguished well to brickpox G4T10 plants in triple vaccine and swine plague EO630 plants, the content of brickpox bacterium is set to be determined accurately, it is reproducible, the degree of accuracy is high, foundation is provided to monitor vaccine quality stabilization in production, is also beneficial to be monitored in buying the quality of vaccine so as to ensure immune effect in place.
Description
Technical field
The invention belongs to the micro-organisms field of animal medicine, a kind of detection swine fever brickpox swine plague is specifically disclosed
Selective medium of trigeminal live vaccine antigen bacterial content and its preparation method and application.
Background technology
Brickpox (Swine erysipelas, SE) is by bacillus rhusiopathiae suis (Erysipelothrix
Rhusiopathiae, ER) a kind of acute febrile infectious disease for causing, it is mainly characterized by hyperpyrexia, acute sepsis, skin rash
Block (subacute), chronic libman-Sack endocarditis and cutaneous necrosis and multiple non suppurative arthritis (Wood R.L.,
Erysipelas,in:Leman,et al.(Eds.),Diseases of swine,Iowa State University
Press,Ames,Iowa,1992,pp.475–486).The bacillus rhusiopathiae suis being separated to from the pig body of these symptoms is mostly blood
Clear 1 type (1a hypotypes and 1b hypotypes) and 2 types (Takashi et al., Protection Effect of NaOH-Extracted
Erysipelothrix rhusiopathiae J.Vet.Med.Sci.60(1):9-14.1998).This disease is once referred to as me
One of state's " three big infectious diseases ", in recent years, as pig intensivization development brickpox gradually fades out the visual field of people, but the disease is simultaneously
It is not cleaned completely, there is fragmentary appearance in area always throughout the country, and serious economic loss (Gong Ping is caused to pig farmer
Sun, Wang Lian thinks that the characteristics of incidence and comprehensive measures for the prevention and control [J] poultry industries of positive will perfume In Guangdong Provinces pig farm brickpox are total, 2010
(11)：11-12.).
Prevent the disease at present mainly carries out immune prevention and control, the vaccine that China uses using bacillus rhusiopathiae suis attenuated vaccine
Predominantly brickpox attenuated vaccine strain G4T10 plants, the vaccine strain is existed by Jiangsu Province Agriculture Science Institute and Nanjing biologics factory
20 century 70 joint research and developments are formed, brickpox low virulent strain G4T10 be velogen strain through the generation of cavy 370 and containing 0.01%~
0.04% acridine yellow blood agar culture-medium uploads what 10 generations obtained.
In order to simplify multiple vaccine injection, in the case where China Veterinary Drugs Supervisory Inst. is presided over, East China Institute of agricultural sciences, Chinese agriculture
Participate in crude drug factory (1974-1978) year such as industry academy of sciences Harbin veterinary institute, Nanjing, Chengdu, Zhengzhou and Lanzhou grinding jointly
It has been made swine fever, brickpox, swine plague trigeminal live vaccine.Bacterium used, strain are brickpox G4T10 plants, pig in combined vaccine
Lung epidemic disease EO630 and hog cholera lapinised virus.Three kinds of bacterium, venom dispense lyophilized forming after mixing by a certain percentage.Also dependent on reality
Epidemic prevention blood medicine prepares Combined vaccine.The immunity for resisting each cause of disease produced by connection seedling immunoprophylaxis pig, draws with each single seedling inoculation
The immunity for rising is basically identical in intensity, and nothing interferes phenomenon between illustrating three.And this laboratory previous experiments knot
Fruit display triple vaccine immunity energy resists the attack of swine fever and brickpox Wen Shi separation strains respectively.
At present, swine fever brickpox swine plague trigeminal live vaccine is commonly used in production to enter three kinds of diseases of swine fever brickpox swine plague
The immune prevention and control of row, contribution of the swine fever brickpox swine plague trigeminal live vaccine to aquaculture small can not be peeped, and its immune effect is directly closed
It is the interests of numerous raisers.Known, the most basic determinant of the immune effect of vaccine is that Effective Antigens contain in vaccine
Amount, but a kind of effective method can well not monitored to brickpox antigen in vaccine in the prior art.
As can be seen here, there is very big deficiency in prior art.
The content of the invention
In view of this, it is necessary to for above-mentioned problem, there is provided one kind detection swine fever brickpox swine plague trigeminal live vaccine
Selective medium of antigen bacterial content and preparation method thereof, and the selective medium is applied to the side of test in laboratory
Method；The selective medium and detection method brickpox and swine plague in commercialization swine fever brickpox swine plague triple vaccine mix
In the case of together, it is still able to carry out Accurate Determining to brickpox antigen bacteria, reproducible, the degree of accuracy is high, in production
Monitoring vaccine quality stabilization provides foundation.
To reach above-mentioned purpose, the present invention uses following technical scheme：
A kind of selective medium for detecting swine fever brickpox swine plague trigeminal live vaccine antigen bacterial content, including following group
Point：TSA (tryptose soya agar) 29-40mg/ml, cow's serum 3%~5%, kanamycins 20~80 μ g/mL are aseptic double
Steam appropriate amount of water.
Further, the selective medium, including following component：TSA (tryptose soya agar) 30mg/ml,
Cow's serum 5%, the μ g/mL of kanamycins 50, appropriate aseptic double-distilled water.
Further, the preparation method of the selective medium includes：Weigh TSA powder and be dissolved in appropriate sterilizing distilled water
In, in 121 DEG C of 16~21min of high pressure, treat that it is cooled to 45-55 DEG C naturally, cow's serum and kanamycins are added, stirring is
Can.
Further, the preparation method of the selective medium includes：Weigh TSA powder and be dissolved in appropriate sterilizing distilled water
In, in 121 DEG C of high pressure 16min, treat that it is cooled to 50 DEG C naturally, cow's serum and kanamycins are added, stirring is equal.
Further, the selective medium, the antigen in Accurate Determining swine fever brickpox swine plague trigeminal live vaccine
Application in bacterial content.
Further, the selective medium is applied to determine brickpox in swine fever brickpox swine plague trigeminal live vaccine
The method of antigen bacterial content, including following operation：
1) got the raw materials ready by the proportioning, selective medium is prepared with the preparation method, it is stand-by；
2) sample process to be checked：Sample triple vaccine SPSS is diluted to 1mL/ part, 100 μ L dilutions are taken
In 900 μ L SPSSs；Aforesaid operations are repeated, serial dilution 6 times takes last time dilution (the i.e. the 6th pipe) 100 μ L
Be inoculated in step 1) prepare selective agar plate in, be placed in 37 DEG C culture 24~48h；
The sample triple vaccine concentration is (3-20) × 108It is 30~200 to grow clump count on 6 rear plates of dilution, is
It is easy to the optimal clump count of accurate counting.
3) to step 2) obtained by bacterium colony count；
Swine fever and brickpox antigenic content are counted with ordinary culture medium by existing method in the triple vaccine.
Further, step 2 described in the detection method of the simply connected vaccine or triple vaccine antigen) in incubation time be
36h。
Further, brickpox antigen is brickpox G4T10 plants of antigen strain in the triple vaccine；The swine plague antigen is
Swine plague is EO630 plants.
Beneficial effect of the present invention：
The selective medium that the present invention is provided, constitutes component and simple structure, prepares convenient easy to operate；It is increased to block that
Mycin can suppress the growth of the growth without G4T10 plants of brickpox of suppression of swine plague EO630 in triple vaccine such that it is able to respectively
Two kinds of bacterial antigens contents of brickpox and swine plague in Accurate Determining triple vaccine；Meanwhile, kanamycins can also prevent some common
The interference of bacterium.The assay method of the selective medium that the present invention is provided is to brickpox G4T10 plants in triple vaccine and swine plague
EO630 plants carries out Accurate Determining respectively, and reproducible, the degree of accuracy is high, and foundation is provided to monitor vaccine quality stabilization in production,
Be conducive to being monitored in buying the quality of vaccine so as to ensure immune effect in place.
Brickpox bacterium and swine plague bacterium mix in commercialization swine fever brickpox swine plague triple vaccine, use Nostoc commune Vanch
Base culture swine plague bacterium meeting Reverse transcriptase brickpox bacteria growing, therefore swine fever and swine plague bacterium can only be counted, it is impossible to it is right
Brickpox bacterium carries out accurate counting.Present invention discover that and using swine plague fast growth and colonial morphology it is big compared with brickpox this
One characteristic, there is provided described selective medium and the detection method pig in commercialization swine fever brickpox swine plague triple vaccine are red
In the case that poison and swine plague are mixed, it is still able to carry out Accurate Determining to brickpox antigen bacteria, it is reproducible, accurately
Degree is high, and foundation is provided to monitor vaccine quality stabilization in production.Cannot be by two kinds of bacterial antigens point in triple vaccine before compensate for
The defect not counted, this is also solved carries out the long-standing problem of quality monitoring in Clinical practice to triple vaccine.
Brief description of the drawings
Fig. 1 is to count situation after being inoculated with plain agar plate after swine plague EO630 unit price seedlings dilute.
Fig. 2 is to count situation after being inoculated with plain agar plate after brickpox G4T10 unit price seedlings dilute.
Fig. 3 is inoculation plain agar plate after being diluted after brickpox G4T10 unit price seedlings and swine plague EO630 unit price seedling mixing
After count situation.
The selective agar of inoculation is put down after Fig. 4 dilutes after mixing for brickpox G4T10 unit price seedlings and swine plague EO630 unit price seedlings
Situation is counted after ware.
Specific embodiment
In order to problem solved by the invention, the technical scheme for being used and the effect for being reached is better described, now tie
Close specific embodiment and related data is expanded on further.It should be noted that present invention is including but not limited to following implementation
Example and combinations thereof implementation method.
If not specializing, the conventional hand that technological means used is well known to the skilled person in embodiment
Section, agents useful for same is commercial goods, and cow's serum is purchased from Wuhan Sanli Ltd., and TSA is purchased from U.S. company BD, and kanamycins is purchased from
Sigma companies；Swine fever brickpox swine plague trigeminal live vaccine, brickpox G4T10 unit price live vaccines, swine plague EO630 unit prices are living
Vaccine is purchased from Zhongmu Industry Co., Ltd.
The Test of accuracy of embodiment 1
(1) preparation of agar plate：
It is prepared by plain agar plate：Weigh 30gTSA powder to be dissolved in 1000mL sterilizing distilled waters, in 121 DEG C of high pressures
16min, treats its cool to 50 DEG C, adds 5% cow's serum；
It is prepared by selective agar plate：Weigh 30gTSA powder to be dissolved in 1000mL sterilizing distilled waters, in 121 DEG C of high pressures
16min, treats its cool to 50 DEG C, adds 5% cow's serum and 30mg kanamycins.
(2) by brickpox G4T10 unit price live vaccines, swine plague EO630 unit price live vaccines are dilute with SPSS respectively
1mL/ part is interpreted into, and takes 100 μ L respectively and be diluted in 900 μ L SPSSs, and be carried out continuously respectively 6 times, taken respectively
100 μ L are inoculated in the plain agar plate without kanamycins in 6th dilution, are placed in 37 DEG C of culture 36h, and bacterium colony is entered
Row is counted.
(3) by brickpox G4T10 unit price live vaccines, SPSS is used after swine plague EO630 unit price live vaccine mixing
It is diluted to respectively containing brickpox and swine plague 1mL/ part, and takes 100 μ L and be diluted in 900 μ L SPSSs, continuously
Carry out 6 times, and take that 100 μ L in the 6th dilution are inoculated in the plain agar plate without kanamycins and that is mould containing card respectively
In the selective agar plate of element, 37 DEG C of culture 36h are placed in, and macrocolony very little colonial morphology bacterium colony in common plate is distinguished
Appraisal counting is carried out, and all bacterium colonies of selective agar are counted.
Differentiate foundation：Swine plague bacteria growing speed is fast compared with brickpox bacterium in ordinary culture medium, and its colonial morphology is larger, directly
Footpath be 3-4 μm, and brickpox then small diameter be 1-2 μm；And in selective medium, swine plague bacterium is suppressed substantially, no
It can be seen that.
(4) count results：As shown in table 1, the count results of step (2) are respectively 117 and 70, show brickpox G4T10
Univalent live vaccine brickpox bacterial content is 11.7 × 108, swine plague EO630 unit price live vaccine bacterial contents are 7 × 108；Step
(3) common plate macrocolony number is 69 in, and petite number is 90, and swine plague is thin in showing former swine plague EO630 unit prices live vaccine
Bacterial content is 6.9 × 108, brickpox bacterial content is 9 × 10 in showing former brickpox G4T10 unit prices live vaccine8, in step (3)
Selective agar count results are 113, show in former brickpox G4T10 unit price live vaccine brickpox bacterial content be 11.3 ×
108, as a result show that plain agar plate can be with swine plague antigenic content after Accurate Determining mixing, and brickpox antigenic content is then needed
Counted in selective plate.Testing result is as shown in Figure 1, Figure 2, Figure 3 and Figure 4.
The experimental data of 1 embodiment of table 1
The accuracy of major embodiment selective medium of the present invention and method in this example, (selects same by same producer
One producer is to ensure the uniformity of antigenic content) the corresponding brickpox list seedling of triple vaccine and swine plague list seedling count respectively
The as two kinds actual contents of single seedling, then we are artificially by the brickpox list seedling and swine plague list of the two known antigens contents
Seedling mixes, and is contrasted with the method for counting of the method for invention and routine respectively, and comparing result can be seen that
The method of invention is consistent, i.e., selective medium of the present invention and detection method institute with the actual content of two kinds of single seedlings
The amount of each single antigen in the polyvalent vaccine for detecting is closest to actual content.
The Repeatability checking of embodiment 2
(1) by embodiment 1 the step of (1) prepares plain agar plate and selective agar plate, by swine fever brickpox pig
Lung epidemic disease triple vaccine is diluted to 1mL/ part, and takes 100 μ L and be diluted in 900 μ L SPSSs, is carried out continuously 6 times, and point
100 μ L are inoculated in the plain agar plate without kanamycins and the selective fine jade containing kanamycins in not taking the 6th dilution
In fat plate, 37 DEG C of culture 36h are placed in, and macrocolony very little colonial morphology bacterium colony in common plate is counted respectively, with
And all bacterium colonies of selective agar are counted, macrocolony number is as swine plague antigenic content, selectivity in common plate
Clump count is brickpox antigenic content in plate.2 repetitions are carried out by aforesaid operations.
(2) result for repeating twice is contrasted, is repeated one：Macrocolony number is 78, petite in plain agar plate
Number is 76, then swine plague antigenic content is 7.8 × 10 in triple vaccine8, brickpox antigenic content is 7.6 × 108, selective agar
Clump count is 108 in plate, then brickpox antigenic content is 10.8 × 10 in triple vaccine8；Repeat two：It is big in plain agar plate
Clump count petite number 72, then swine plague antigenic content is 7.2 × 10 in triple vaccine8, brickpox antigenic content is 8.3 × 108,
Clump count is 112 in selective agar plate, then brickpox antigenic content is 11.2 × 10 in triple vaccine8.Testing result is shown in Table
2。
The measuring samples testing result of table 2
Result shows to repeat identical incubation time twice, because brickpox colony growth rate is grown slowly in common plate
Fireballing swine plague bacterium colony covering is repeated bad and inaccurate so as to cause common plate to count brickpox antigenic content,
And selectivity agar plate is then accurate and reproducible.
The detection of the different lot number swine fever brickpox swine plague trigeminal live vaccines of embodiment 3 pairs
Test group：To three swine fever brickpox swine plague trigeminal live vaccines of different batches respectively according to the embodiment of the present invention
Step (1) method is diluted in 2, inoculation, and then culture counts, and macrocolony number is as swine plague antigen in common plate
Content, clump count is brickpox antigenic content in selective plate.
Control group：The step of by embodiment 1 (1), prepares plain agar plate and selective agar plate, and swine fever pig is red
Malicious swine plague triple vaccine is diluted to 1mL/ part, and takes 100 μ L and be diluted in 900 μ L SPSSs, is carried out continuously 6 times,
And 100 μ L are inoculated in the plain agar plate without kanamycins with taking the 6th dilution respectively, 37 DEG C of culture 36h are placed in,
And macrocolony very little colonial morphology bacterium colony in common plate is counted respectively, macrocolony number is as pig lung in common plate
Epidemic disease antigenic content, small clump count is brickpox antigenic content in common plate.
Above-mentioned two groups of experimental results are as shown in table 3：
Bacterial antigens assay result in the different batches triple vaccine of table 3
As can be seen that the method for the present invention is to swine fever brickpox swine plague trigeminal live vaccine bacterial antigens content in table 3
The mensuration mode compared with prior art is determined closer to actual content, i.e., it is more accurate.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Shield scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (8)
1. a kind of selective medium for detecting swine fever brickpox swine plague trigeminal live vaccine antigen bacterial content, including following group
Point：TSA29-40mg/ml, cow's serum 3%~5%, the μ g/mL of kanamycins 20~80, appropriate aseptic double-distilled water.
2. selective medium according to claim 1, it is characterised in that including following component：TSA30mg/ml, ox blood
Clear 5%, the μ g/mL of kanamycins 50, appropriate aseptic double-distilled water.
3. it is a kind of detect swine fever brickpox swine plague trigeminal live vaccine antigen bacterial content selective medium preparation method, its
It is characterised by, including：Weigh TSA powder to be dissolved in appropriate sterilizing distilled water, in 121 DEG C of 16~21min of high pressure, treat that it puts naturally
Cool to 45-55 DEG C, cow's serum and kanamycins are added, stirring is equal.
4. the preparation method of selective medium according to claim 3, it is characterised in that including：Weigh TSA powder molten
In appropriate sterilizing distilled water, in 121 DEG C of high pressure 16min, treat that it is cooled to 50 DEG C naturally, add cow's serum and kanamycins,
Stirring is equal.
5. the selectivity described in the selective medium or claim 3 or 4 described in claim 1 or 2 prepared by preparation method
Culture medium, the application in antigen bacterial content in determining swine fever brickpox swine plague trigeminal live vaccine.
6. a kind of method for detecting swine fever brickpox swine plague trigeminal live vaccine brickpox antigen bacterial content, including following operation：
1) selective medium described in claim 1 or 2 is prepared with preparation method described in claim 3 or 4, it is stand-by；
2) sample process to be checked：Sample triple vaccine SPSS is diluted to 1mL/ part, 100 μ L is taken and is diluted in
In 900 μ L SPSSs；Aforesaid operations are repeated, serial dilution 6 times takes last time dilution (the i.e. the 6th pipe) 100 μ L and connects
Kind in step 1) in the selective agar plate for preparing, it is placed in 37 DEG C of 24~48h of culture；
3) to step 2) obtained by bacterium colony count.
7. detection method according to claim 6, it is characterised in that the step 2) in dilution number of times be 6 times；Culture temperature
Spend is 37 DEG C；Incubation time is 36h.
8. the detection method according to claim 6 or 7, it is characterised in that the brickpox antigen bacteria in the triple vaccine
It is brickpox G4T10 plants of antigen strain.
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| CN113046409A (en) * | 2021-03-22 | 2021-06-29 | 中海生物技术(枣庄)有限公司 | Selective chromogenic medium for colony of erysipelothrix suis and pasteurella multocida |
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